Biology Reference
In-Depth Information
immunodetection with specific antibodies, extra washing steps are recommended.
Also, increasing the salt concentration in the RIPAwashing buffer will make the con-
ditions more stringent and can help to remove proteins nonspecifically bound to the
beads. After this procedure, only proteins recognized by the used antibody and the
interacting partners should stay attached to the beads.
17.2.3.2
Membrane coimmunoprecipitation: on living cells
(Fig. 17.2A, right)
Membrane co-IP is a modification of the standard co-IP we designed to isolate pro-
teins from the plasma membrane together with the interacting partners.
Cells (transfected or from native tissue) are collected in PBS (250 g, 4
C, 10 min)
and resuspended in 1 ml of serum-free medium. Primary antibody recognizing the ex-
tracellular epitope of the receptor is added to the cells in the dilution previously op-
timized for each specific antibody (examples of optimized dilutions of antibodies
frequently used in our laboratory can be found in
Table 17.5
). We prefer to work at
4
C to prevent receptor internalization but incubation at 37
C is also possible. After
2 h, cells are spun down (250
g, 4
C, 10 min) and washed once with PBS, and stan-
dardRIPA lysis protocol starts (see
Section 2.2.2.1
). Upon clearing the lysates, Protein
A beads are immediately added and overnight incubation at 4
C with rotation is per-
formed. By using this modification of IP, it is possible to isolate only mature receptors
present at the plasma membrane, and one can be sure that the observed interaction oc-
curs with fully processed receptors and not in the ER or Golgi apparatus.
17.2.4
Elution
After removing all proteins that are unbound or nonspecifically bound to the beads,
the elution procedure can start. Different methods allowing elution of proteins from
the beads exist, but most often, SDS buffer is used as it provides the highest elution
Table 17.5
Examples of Optimized Dilutions of Primary Antibodies Used for mco-IP
and Immunoblotting
Catalog
Number
Dilution,
mco-IP
Dilution,
IB
Antibody
Company
Anti-HA.11 (16B12)
Covance
MMS-
101P-1000
1:800
1:2000
Anti-HA rabbit
GeneTex
GTX29110
Not tested
1:2000
Anti-FLAG (M2)
Sigma
F3165
1:800
1:4000
Anti-c-myc (4A6)
Millipore
05-724
Not tested
1:2000
Anti-mOR (384-398)
Calbiochem
PC165L
Recognizes
intracell. loop
1:2000
Anti-D
4
R (D16)
Santa Cruz
Biotechnology
Sc-31481
Not tested
1:100
For standard co-IP we always add 2 mg antibody per sample.
